Enzyme Linked Immunosorbent Assay

Excerpt

Enzyme immunoassays (EIAs) use the catalytic properties of enzymes to detect and quantify immunologic reactions. Enzyme-linked immunosorbent assay (ELISA) is a heterogeneous EIA technique used in clinical analyses. In this type of assay, one of the reaction components is nonspecifically adsorbed or covalently bound to the surface of a solid phase, such as a microtiter well, a magnetic particle, or a plastic bead. This attachment facilitates the separation of bound and free-labeled reactants.

In the most common approach to using the ELISA technique, an aliquot of sample or calibrator containing the antigen (Ag) to be quantified is added to and allowed to bind with a solid-phase antibody (Ab). After washing, an enzyme-labeled antibody is added and forms a “sandwich complex” of solid-phase Ab-Ag-Ab enzyme. Unbound antibody is then washed away, and enzyme substrate is added. The amount of product generated is proportional to the quantity of antigen in the sample.

Specific antibodies in a sample can also be quantified using an ELISA procedure in which antigen instead of antibody is bound to a solid phase. The second reagent is an enzyme-labeled antibody specific to the analyte antibody. In addition, ELISA assays have been used extensively to detect antibodies to viruses and autoantigens in serum or whole blood. In addition, enzyme conjugates coupled with substrates that produce visible products have been used to develop ELISA-type assays with results that can be interpreted visually. Such assays are very useful in screening, point-of-care, and home testing applications.